Collagens belong to an ever-expanding family of proteins that have the capacity to form extracellular fibrils or network-like structures and fulfill a variety of essential biological functions in vertebrates (Fleischmaier et al. (1990) Ann. N.Y. Acad. Sci. 580:161-175; Engel and Prockop (1991) Annu. Rev. Biophys. Biophys. Chem. 20:137-52; Prockop and Kivirikko (1995) Annu. Rev. Biochem. 64:403-34). Structurally, they are characterized by the presence of several repeats of the amino acid sequence -Gly-X-Y-(where X=proline and Y=4-hydroxyproline). Additionally, they have the potential for the generation of three chains with such repeated sequences that fold into a characteristic triple helix. This assembly and characteristic folding conformation influences the ability of the collagens to polymerize. A correctly assembled triple helix is relatively rigid which is important for the biological function of the protein.
The most abundant types of collagen in the body are those that form fibrils (types I, II, III, V, and XI) (Hulmes (1992) Essays Biochem., 27:49-67). Type I collagen has the most extensive distribution and is consequently the most abundant of the fibril-forming collagens. It is present in most connective tissues. Type II collagen is the second most abundant collagen but has a somewhat more selective tissue distribution, being found in both the cartilage and vitreous humor of the eye. Due to the widespread distribution and essential function of these collagens, over 300 different mutations in fibril collagen genes have been identified thus far in patients afflicted with a variety of diseases.
The elucidation of mutations in human patients through familial genetic analysis has identified over 50 alterations alone in the gene for collagen type II (COL2A1), the most abundant protein in cartilage (Kuivaniemi et al. (1991) FASEB J., 5:2052-60; Kivirikko (1993) Ann. Med., 25:113-26). These mutations in the COL2A1 gene cause a spectrum of cartilage defects ranging from moderate phenotypes to lethal forms of chondrodysplasia and skeletal deformities (Vikkula et al. (1994) Ann. Med., 26:107-14). Mutations in COL2A1 are also found in approximately 2% of patients with early onset familial osteoarthritis (OA) (Ritvaniemi et al. (1995) Arthritis Rheum., 38:999-1004). Specific mutations found in patients with OA include a substitution of cysteine for arginine at amino acid 519 of the alphal (II) chain, and serine for glycine mutations at positions alphal-274 and alphal-976 (Williams et al. (1994) Matrix Biol., 14:391; Ala-Kokko et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6565-68; Spranger et al. (1994) Eur. J. Pediatr., 153:56-65).
Several studies have been performed using transgenic mice in which mutated collagen genes were randomly inserted to produce dominant negative effects. Additionally, studies have been carried out involving the inactivation of collagen genes either by viral insertion (Bonadio et al. (1990) Proc. Natl. Acad. Sci. USA, 87:7145-49) or by knock-out of collagen genes or portions of collagen genes (Li et al. (1995) Genes and Dev., 9:2821-30). Studies with transgenic animal models have shown that mutated human COL2A1 gene constructs can cause phenotypes similar to chondrodysplasia seen in human patients with dwarfism, a short snout, a cranial bulge, cleft palate, and delayed mineralization of the bone (Vandenberg et al. (1991) Proc. Natl. Acad. Sci. USA, 88:7640-44). Expression of specifically mutated mouse COL2A1 genes resulted in similar phenotypes of severe chondrodysplasia (Nakata et al. (1991) Proc. Natl. Acad. Sci. USA, 88:9648-52; Metsaranta et al. (1992) J. Cell Biol., 118:203-12). In older mice from the same lines, the evidence of chondrodysplasia was less marked, and the most striking features were degenerative changes of articular cartilage similar to osteoarthritis (Helminen et al. (1993) J. Clin. Invest., 92:582-95). Overexpression of a normal mouse COL2A1 gene in transgenic mice produced abnormally thick collagen fibrils in cartilage, apparently because of an imbalance in the amounts of collagen being synthesized in the tissues (Garofalo et al. (1993) Proc. Natl. Acad. Sci. USA, 90:3825-29).
An experimental approach for studying the physiological effects of mutations on collagen function that has met with some success is introduction of a gene that encodes one polypeptide component of collagen into animals that express the other component. The transferred gene may also be engineered to carry defined mutations. The endogenous gene corresponding to the transferred gene is necessarily functionally deleted with no effect on the other subunit component.
Wu et al. (1990) Mol. and Cell Biol., 10:1452-60, used this approach to show that a human-mouse interspecies collagen I heterotrimer does form and is functional in embryonic development of transgenic mouse embryos. However, rescue of the phenotype is only partial, since embryos die soon after birth. Similarly, Vandenberg et al. (1991) Proc Natl. Acad. Sci. USA, 88:7640-44 prepared transgenic mice expressing a minigene version of the human COL2A1 gene along with the mouse gene. In cultured chondrocytes prepared from the transgenic mice, the minigene was expressed as shortened pro-alphal (II) chains that were disulfide-linked to normal mouse pro-alphal (II) chains. It was suggested that the presence of the shortened pro-alpha chain in a procollagen molecule can prevent folding into a stable triple helix that results in degradation of normal genes in a phenomenon known as procollagen suicide.
de Crombrugghe et al. (1995) J. Rheumatol., 22:1 Supp. 43:140-142 generated transgenic mice by introducing dominant negative mutations in the mouse COL2A1 gene. Mice homozygous for the mutant transgene died at birth but showed a phenotype of severe chondrodysplasia with skeletal anomalies. Electron microscopic analysis revealed an absence of normal collagen fibrils in cartilage. Mice heterozygous for the transgene, however, showed no significant abnormalities at birth but developed clear signs of osteoarthritis with erosion of cartilage structure in joints by six to nine months of age. Severity of the phenotype was linked to expression of the mutant gene. Thus, transgenic animals, if engineered with specific mutations, provide a useful system to understand the pathoetiology of type II collagen diseases, particularly, OA and to evaluate therapeutic drugs for blocking degenerative changes in cartilage.
Transgenic mice capable of expressing the human collagen gene and not the mouse collagen gene have now been developed. These mice are especially useful in the development of compositions and methods of treating human type II collagen related diseases. Furthermore, the chondrocytes, the cells that synthesize procollagen type II from the transgenic animals that express the normal human COL2A1 gene, provide a source of biomaterial to treat cartilage related diseases.
An object of the present invention is to produce a transgenic mouse model system capable of expressing the human collagen gene wherein the endogenous mouse collagen gene is inactivated.
Another object of the present invention is the utilization of the transgenic mouse model system to develop compositions and methods for the treatment of cartilage related disorders.
Yet another object of the present invention is the production of quantities of biomaterials for the treatment of cartilage related disorders.